374A AASLD ABSTRACTS HEPATOLOGY October 1995

1069 ORAL CALCIUM PHOSPHATE LOWERS SERUM BILIRUBIN LEVELS IN CRIGLER-NAJJAR TYPE I, BUT NOT IN TYPE II PATIENTS C.N. van tier Veere, P.L.M. Jansen. M. SinaasaDDel, R. van der Meet, H. van

de Si/s, J.A. Rammeloo, P.H. Goyens, CM.J. van Nieuwkerk, R.P.J. Oude Elferink. Dept. of Gastroenterology and Dept. of Pharmacy, Academic Medical Center Amsterdam; Sophia Children Hosp. Rotterdam; Dept. of Nutdtion, Netherlands Inst. for Dairy Res. Ede; Elizabeth Hosp. Tilburg; The Netherlands, and Queen Fabiola Hosp. Brussels, Belgium.

Cdgler-Najjar disease, hereditary unconjugated hyperbilirubinemia, is caused by a complete (type I) or partial (type II) deficiency of bilirubin-UDP- glueuronosyltransferase in the liver. Gunn rats are the animal model for this disease, Intestinal disposition may be a prominent excretion pathway of unconjugated bilirubin (UCB) in these patients and animals, Previously we have shown that a high calcium phosphate content of the diet leads to a decrease in serum bilirubin in Gunn rats, probably by binding of UCB to precipitated calcium phosphate in the intestine (C.N van der Veere et al., Hepatology 1993, vol,18:127A). The effect of oral calcium phosphate on plasma bilirubin was evaluated in eleven patients with Crigler-Najjar disease, aged 2 to 42 years. Five patients were diagnosed as type I, and six patients as type II. Methods: A double-blind, placebo controlled, cross-over design was used. The usual therapeutic management (phototherapy and/or phenobarbital) was continued. In adults 100 mmol calcium and in children 2.5 mmol/kg bodyweight (50% CaCO3 and 50% CaHPO4.2H20)was orally administered in the treatment period. Cellulose was used as a placebo. Results: Bilirubin levels decreased significantly in the type I patients (17.9±6.4%, p=0.03) but not in the type II patients in the treatment pedod. No differences were found in plasma Ca, Mg, P, Hb, Htc, LDH, urea, creatinine, and phenobarbital levels. Urinary output of calcium and phosphate was similar in the treatment and reference pedod, which reflects the lack of resorption of both compounds from the gut. The effect of calcium phosphate in CN patients correlated with the intensity of phototherapy. Conclusions: We propose that calcium phosphate enhances the efficacy of phototherapy by intestinal binding of UCB that was secreted as bilirubin photoisomers into bile but reverted to UCB in the intestine. In the absence of calcium phosphate UCB can be reabsorbed from the gut. Oral calcium phesphate may become a useful adjuvant to photo therapy in Cdgler-Najjar type I disease and might diminish the need for phototherapeutic treatment.

1070 CORRECTION oF BIURUBIN GLUCURONIDATION DEFICIENCY IN GUNN RATS BY TRANSPLANTATION OF GUNN RAT FIBROBLASTS 'EXPRESSING BILIRURIN UDP-GLUCURONOSYLTRANSFERASE. J,Seonen. K.Tada#. S.Hellwia. J.Rov Chowdhurv#. N.Rov ~:howdhurv#. P.J.Bosma, and R.P.J.Oude Elferink Academic Medical Centre, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands, # Albert Einstein College of Medicine, New York, USA.

Introduction: Patients with Crigler-Najjar syndrome {CN) and its animal model, the Gunn rat, have an inherited deficiency of the hepatic enzyme bilirubin UDP-glucuronoayltransferase (B-UGT). As a result, they accumulate toxic serum levels of unconjugated bilirubin. We have explored the possibility of extrahepetic gene therapy by transplantation of autologous fibroblaats which expressed B-UGT. Methods: Primary Gunn rat fibroblests (GURF) were transduced with a recombinant retrovirus, capable of transferring B-UGT cDNA and a ceil line with high expression of B-UGT was cloned (GURF-B1). The GURF were transplanted in the peritoneal cavity of syngeneic Gunn rats. Serum bilirubin of the treated rats was measured and bile was analysed with HPLC, Results: The B- UGT activities in homogenates of GURF-B1 and freshly isolated Wistar hapatocytss were comparable, 2.9 ± 0.3 and 3 .4± 0.2 nmol/h*mg protein respectively. Bilirubin added to the culture medium of GURF-B1 was glucuronidated and excreted, The B-UGT activities of intact GURF-BI and intact freshly isolated Wistar hepatocytea were comparable when physiologic bilirubin concentrations (5-20 #M) were present in the culture media. At 40-80 pM bilirubin, the hapatocytas were more active than GURF-B1. When 3.10 s GURFoB1 were transplanted in Gunn rats serum bilirubin dropped from 116±8 ( t=0) to 53,+7 pM I t = 4 daysl,(n=3). Seven days after transplantation of GURF-B1 serum bilirubin had dropped to 3 6 ± 3 pM (n=2). The serum bilirubin of control Gunn rats which received the same amount of untranafected GURF remained constant, 113±10 ( t=0 l 9 9 ± 1 8 #M (tffi4 days),(n~3), Four days after transplantation, bilirubin conjugates were detected in bile of Gunn rats which received,the GURF-B1, but not in bile of control Gunn rats. Conclusions: Expression of B-UGT in fibroblasts enables them to perform all metabolic steps necessary for the glucuronidation of bilirubin. Fibroblasts expressing B-UGT could be used to develop extrahepatic gene therapy for CN disease.

1 0 7 1 LONG-TERM CORRECTION OF BILIRUBIN GLUCURONIDATION DEFECT IN GUNN RATS BY EX VIVO GENE THERAPY USING CONDITIONALLY IMMORTALIZED AUTOLOGOUS HEPATOCYTES. K Tada. N Roy ChowdhuD'. VP, Prasad 1, P Menchikalanudi. IJ Fox:, J ~.eppen 3. PJ Bosn~3. J Roy Chowdhurv. Dept, of Medicine, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY; :Dept. of Transplantation SurgeD,, Uin')ersity of Nebraska, Omaha, NE; 3Dept. of Gastroenterology and Liver Diseases, Academic Medical Center, Amsterdam, The Netherlands.

Ex viyo gene therapy, in which hepatocytes are harvested from mutants, retrovirally transduced with a normal gane and transplanted back into the donor, has been used for correction of inherited metabolic defects of the liver (Science 254:1802-1805, 1991). Major drawbacks of this method include limited availability of primary hepatoeytes and inefficient retroviral transduction as primary hepatocytes do not actively divide in culture. To obviate these problems, we immortalized primary hepatoeytes derived from inbred bilirubin-UDP- gluenronosyltransferase (B-UGT) deficient Gunn rats (an animal model for Crigler-Najjar syndrome type I) by infection with a recombinant retrovims • expressing a temperature-sensitive mutant SV40 large T antigen (~T). The cloned immortalized cells were than transduced with a second recombinant retrovirus expressinghtanan B-UGT~andacloneexpressinghighlevelsoftheenzyme was expanded by culturing at permissive temperature (33°C). At 37°C, the ~T antigen was degraded and the cells stopped dividing. Immunoblot studies showed that these cells expressed human B-UGT~ and UGT activity toward bilirubin was twice that of normal human liver homogenates. Following intrasplenic injection of 2x107 of these autologous cells into Gurm rats (n=7), excretion of bilirubin glueuronides in bile was demonstrated by HPLC analysis and serum bilirubin levels were reduced by 15 to 25% in two weeks and remained So throughout the duration of the study (50 days). None of the transplanted Gunn rats or SCID mice transplanted with the immortalized cells developed tumors. CONCLUSION: Autologous transplantation of mutant hepatocytes following conditional immortalization, expansion in culture and transduction with a normal gene provides effective ex vivo gene therapy, that may be useful in the treatment of Crigler-Najjar syndrome type I and other inherited enzyme deficiency disorders.

1072 GILBERT SYNDROME IS ASSOCIATED W I T H NEONATAL JAUNDICE. JD Bancroft and GR Gourley. University of Wisconsin Medical School, Madison, WI.

A molecular marker for Gilbert Syndrome (GS) was recently suggested (Bosma et al, Hepatology 1994, 20:266a). GS is associated with hyperbili- rubinemia and decreased bilirubin UDP-glucuronosyltransferase activity (B-UGT) in adults. However, GS is usually diagnosed after puberty and the role of GS in the neonatal period is unknown. The current study tested the hypothesis that this marker (an extra TA in the TATAA box of the B- UGT gene, UGTIA, promoter) is associated with neonatal jaundice. Study population: 67 normal newborn infants ~ 35 weeks gestation. Methods: Transcutaneous jaundice index was measured shortly after birth and daily for the first week of life (Minolta-Air Shields Jaundice Meter). Fecal bilirubin diglucuronide (BDG) and total bile pigments were measured (HPLC). Genomic DNA was isolated from blood and the UGT1A pro- moter with TATAA box amplified (PCR) yielding 90 [(TA)7 , normal] or 92 [(TA)8, GS] base pair products that were identified (PAGE). Statistical analysis employed Kruskal-Wallis (KW) and Wilcoxon (W) tests. Results: (TA)s homozygotes (n=7) had greater increase in jaundice index from day 0 to 2 (mean±SD:7.8~-l.9) 'b than heterozygotes (n=34) (5.8± 3.2) b or (TA)7 (n=26) (5.3±2.8)' (KW:p<0.036; W:'p<0.008, bp<0.05). Similar significant differences in jaundice index increase were seen from day 0 to 1. Day 4 fecal BDG excretion was lower in (TA)8 (173±61 nmol/g dry stool) than heterozygotes (707±704) or (TA)7 (565±433) (KW:p<0.029). Similar significant differences were present on days 5 and 6. On both day 4 and 5 total fecal bile pigments were lower in (TA)8 than in heterozygotes or (TA)7 CKW: p<0.04 and p<0.02, respectively). Condusion: Newborn infants with the putative molecular marker for GS have an associated increase in neonatal jaundice and decreased fecal excretion ofbilirubin diglucuronide and total bile pigments. GS is a new factor contributing to the multifactorial risk of neonatal jaundice.